Melanoma associated peptide analogues and vaccines against melanoma

ABSTRACT

The present invention is concerned with cancer treatment and diagnosis, especially with melanoma associated peptide analogues with improved immunogenicity, epitopes thereof, vaccines against melanoma, tumor infiltrating T lymphocytes recognizing the antigen and diagnostics for the detection of melanoma and for the monitoring of vaccination. The peptides according to the invention can be exploited to elicit native epitope-reactive Cm. Usage of the peptides with improved immunogenicity may contribute to the development of CTL-epitope based vaccines in viral disease and cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of application Ser. No.09/214,836 filed Oct. 4, 1999, which was the National Stage ofInternational Application No. PCT/EP97/03712 filed Jul. 8, 1997 andhaving a priority date of Jul. 11, 1996. The disclosure of each of theserelated applications is incorporated herein in their entireties.

BACKGROUND OF THE INVENTION

[0002] The present invention is concerned with cancer treatment anddiagnosis, especially with melanoma associated peptide analogues,epitopes thereof, vaccines against and diagnostics for the detection ofmelanoma and for the monitoring of vaccination.

[0003] During the stepwise changes from normal to tumor tissue,tumor-associated antigens appear. The characteristics oftumor-associated antigens are very much dependent on the origin of thetumor carrying them. The existence of antigens associated with animaltumors was documented in the last century, and the antigenic characterof human cancers has been well established, primarily through recentstudies with monoclonal antibodies.

[0004] Attempts to isolate and chemically characterize these antigenshave encountered serious difficulties, many having to do with a lack ofreagents suitable for precipitation of the antigen-bearing moleculesfrom a solution.

[0005] Like many other stimuli, the tumor-associated antigens activatenot one but a whole set of defense mechanisms—both specific andunspecific, humoral and cellular. The dominant role in in vivoresistance to tumor growth is played by T lymphocytes. These cellsrecognize tumor-associated antigens presented to them by antigenpresenting cells (APCs), and will be activated by this recognition, andupon activation and differentiation, attack and kill the tumor cells.

[0006] Cytotoxic T lymphocytes (CTL) recognize short peptide fragmentsof 9-11 amino acids in length, which are presented in theantigen-binding groove of Major Histocompatibility Complex (MHC) class Imolecules (Townsend et al., 1986, Cell 44.959; Bjorkman et al., 1987,Nature 329:512). These peptides are usually derived from intracellularprotein pools and associate in the lumen of the endoplasmic reticulumwith MHC class I heavy chain and β2-microglobulin molecules, followed bytransportation of the MHC-peptide complex to the cell surface. Despitethe presence of many putative antigenic peptides within the sameantigen, only a few peptides are selected for recognition by CTL.

[0007] MHC Class I/II antigens are often down regulated in solid tumors.This may affect all class I/II antigens, or only part of them. Viral andcellular peptides that can sensitize appropriate target cells forcytotoxic T lymphocyte mediated lysis may fail to do so when produced incells with a low level of expression of MHC class I antigen. Cytotoxicsensitivity may be induced, at least in some cases by raising the levelof MHC class I/II antigen expression by interferon γ and tumor necrosisfactor α.

[0008] The MHC class I binding-affinity of an epitope is an importantparameter determining the immunogenicity of the peptide-MHC complex.Analysis of Human histocompatibility antigen (HLA-A *0201)-restrictedepitopes recognized by anti-viral CTL demonstrated that several peptidesbind to HLA-A *0201 with high affinity. Furthermore, immunogenicityanalysis of motif containing potential epitopes using HLA-A *0201transgenic mice revealed that a threshold MHC class I affinity wasrequired for a peptide in order to elicit a CTL response (Ressing etal., 1995, J. Immunol. 154:5934; Sette et al., 1994, J. Immunol.153:5586). In addition to the MHC class I-binding affinity, stability ofpeptide-MHC complexes at the cell surface contributes to theimmunogenicity of a CTL epitope. Consequently, MHC class Ibinding-affinity and stability of peptide-MHC complexes are importantcriteria in the selection of specific peptide determinants fordevelopment of CTL-epitope based therapeutic vaccines.

[0009] Recently, a number of antigens have been identified as targetantigens for anti-melanoma CTL. Using a genetic approach, the tumorspecific antigens MAGE-1 and -3, as well as the melanocyte-lineagespecific antigen tyrosinase, were identified (van der Bruggen et al.,1991, Science 254:1643; Gaugler et al., 1994, J. Exp. Med. 179:921;Brichard et al., 1993, J. Exp. Med. 178:489).

[0010] In the co-owned and co-pending patent-application (EP 0 668 350),the gp100 melanocyte-specific protein was identified as a target antigenfor melanoma tumor infiltrating lymphocytes.

[0011] Recently, two other melanocyte differentiation antigens,Melan-A/MART-1 and gp75, were identified as target antigens foranti-melanoma CTL (Coulie et al., 1994, J. Exp. Med. 180:35; Kawakami etal., 1994, Proc. Natl. Acad. Sci. USA. 91:3515; Wang et al., 1995, (vol181, pg 799, 1995). J. Exp. Med. 181:1261. 10-12). Eight HLA-A *0201restricted epitopes derived from these antigens have now beencharacterized, displaying varying affinities for HLA-A *0201 (Wolfel etal., 1994, Eur. J. Immunol. 24:759; Cox et al., 1994, Science 264:716;Kawakami et al. 1995. J. Immunol. 154:3961; Bakker et al., 1995, Int. J.Cancer 62:97; Kawakami et al., 1994, J. Exp. Med. 180:347; Castelli etal., 1995, J. Exp. Med. 181:363).

DISCLOSURE OF THE INVENTION

[0012] In an attempt to improve the immunogenicity of two HLA-A *0201presented epitopes derived from the melanocyte differentiation antigensgp 100 and Melan-A/MART-1, amino acid substitutions within the epitopesto improve HLA-A *0201-binding affinity were performed.

[0013] Surprisingly, it was found that these epitope-analogues have animproved immunogenicity in view of the original epitope. Furthermore, inthe present invention it is demonstrated that the epitope-analoguesallow the induction of peptide-specific CTL displaying cross-reactivitywith target cells endogenously processing and presenting the nativeepitope.

[0014] Usage of these epitope-analogues according to the presentinvention with improved immunogenicity may contribute to the developmentof CTL-epitope based vaccines in chronic viral disease and cancer.

[0015] In more detail, since MHC class I-affinity and peptide-MHCcomplex-stability are important parameters determining theimmunogenicity of an MHC class I presented epitope, the possibility toimprove the capacity of two melanocyte differentiation antigen-derivedepitopes to bind to HLA-A *0201 without affecting interactions with theT-cell receptor (TCR) is explored. Detailed analysis of theMelan-A/MART-1 27-35 and gp100 154-162 epitopes using alaninesubstitutions revealed that amino acids at positions 4 to 7(Melan-A/MART-1 27-35) or 5 to 7 (gp100 154-162) are critical residuesfor TCR recognition. These data are in line with X-ray crystallographystudies of the HLA-A *0201 molecule (Saper et al., 1991, J. Mol. Biol.219:277; Latron et al., 1992, Science 257:964), implying a role for themore permissive residues at position 4 and 5 of the peptide orientedtowards the outside of the MHC molecule, as prominent TCR contact sites.It is demonstrated that for HLA-A *0201 the amino acids at positions 6and 7 of the Melan-A/MART-1 27-35 and gp100 154-162 epitopes do not onlyinteract with secondary pockets in the MHC peptide-binding cleft, butthat they are also critical residues for TCR interaction (Ruppert etal., 1993, Cell 74:929; Madden et al., 1993, Cell 75:693).

[0016] Surprisingly, the alanine substitution at position 8 in the gp100154-162 epitope, KTWGQYWAV (SEQ ID NO: 1), resulted in a peptide thatdisplayed increased HLA-A *0201 affinity. Moreover, thisepitope-analogue was recognized by gp100-reactive CTL at tenfold lowerconcentrations compared to the native epitope. These data demonstratethat amino acid substitutions at a non-anchor position can result inincreased MHC class I affinity and T cell recognition.

[0017] By N-terminal anchor replacements with V, L, M or I towards theHLA-A *0201 binding-motifs were set out to identify epitope-analoguesfor both epitopes with improved affinity for HLA-A *0201 that were stillrecognized by wild type epitope-reactive CTL. For the Melan-A/MART-1epitope, epitope-analogues were obtained with comparable (M) or improved(V, L and I) affinity for HLA-A *0201. However, all N-terminal anchorreplacements resulted in decreased T cell reactivity. Apparently, incase of this epitope, the N-terminal anchoring residue affects thepositioning of the side chains in the center of the peptide, therebyabrogating TCR interactions. Recently, a similar observation has beendescribed involving an HLA-B*3501 restricted epitope of the influenza Amatrix protein (Dong et al., 1996, Eur. J. Immunol. 26:335).Substitution of a serine residue at position 2 of the peptide for themore common HLA-B*3501 N-terminal anchor proline, considerably enhancedbinding to HLA-B*3501, but the epitope-analogue was not recognized byCTL reactive with the native epitope. Moreover, this peptide behaved asa peptide-antagonist as was demonstrated for T cell recognition of bothMHC class II and class I-presented peptides (Dong et al., 1996, Eur. J.Immunol. 26:335; De Magistris et al., 1992, Cell 68:625; Klenerman etal., 1994, Nature 369:403). These findings illustrate that anchorresidue substitutions not only affect MHC class I binding, but in somecases they may also result in a conformational change of the peptide-MHCcomplex, leading to an altered interaction with the TCR.

[0018] However, in case of the gp100 154-162 epitope, in addition to thealanine substituted analogue KTWGQYWAV (SEQ ID NO: 1), three anchorsubstituted epitope-analogues KVWGQYWQV (SEQ ID NO: 2), KLWGQYWQV (SEQID NO: 3), and KIWGQYWQV (SEQ ID NO: 4), with improvedHLA-A*0201-affinity that were recognized by anti-gp100 CTL at tenfoldlower concentrations compared to the wild type epitope were obtained. Invivo immunization experiments using HLA-A*0201/K^(b) transgenic micedemonstrated that these epitope-analogues were immunogenic, resulting inthe induction of murine CTL reactive with both the epitope-analogues andthe native epitope. The immunogenicity of the epitope-analogues wasexpected since the peptide-MHC complex stability of both theepitope-analogues and the native epitope was comparably high.

[0019] In vitro CTL induction experiments using donor derived PBLdemonstrated that epitope-analogue specific CTL could be obtaineddisplaying cross-reactivity with tumor cells endogenously presenting thewild type epitope. In addition to T lymphocytes reactive with the wildtype epitope, the T cell repertoire of healthy donors apparently alsocontains T cells reactive with the gp100 154-162 epitope-analogues.Analysis of TCR usage of cloned CTL reactive with the different gp100154-162 epitope-analogues and with wild type gp100 154-162 will beinformative of the spectrum of the T cell repertoire that can be used toinduce CTL reactivity towards the wild type epitope. With respect toimmunotherapy of cancer, activation of multiple specificities in the Tcell repertoire against an antigenic tumor epitope usingepitope-analogues may increase the possibility of a patient to mount asuccessful anti-tumor response upon immunization. In addition, modifiedepitopes might still elicit immune responses if tolerance against thewild-type epitope is observed.

[0020] Employment of “improved” epitopes in immunotherapy protocolsincreases the amount of peptide-MHC complexes at the cell surface ofantigen presenting cells in vivo, and will result in enhanced priming ofantigen-specific CTL. Apart from their potential in cancerimmunotherapy, usage of epitope-analogues with improved immunogenicitymay contribute to the development of CTL-epitope based vaccines inchronic viral disease.

[0021] Therefore, the present invention includes peptides, immunogenicwith lymphocytes directed against metastatic melanomas, characterized inthat it comprises at least part of the amino-acid sequence of SEQ ID NO:9 wherein the amino-acid at position 2 or 8 is substituted.

[0022] A preferred embodiment of the present invention are peptides,wherein at position 2 Threonine is substituted by Isoleucine, Leucine orValine.

[0023] Another preferred embodiment of the present invention arepeptides, wherein at position 8 Glutamine is substituted by Alanine.

[0024] A specific preferred embodiment of the present invention arepeptides, characterized in that it comprises the amino-acid sequence ofany of SEQ ID NOS: 1-4 or 32-34.

[0025] The term “peptide” refers to a molecular chain of amino acids,does not refer to a specific length of the product and if required canbe modified in vivo or in vitro, for example by manosylation,glycosylation, amidation, carboxylation or phosphorylation: thus interalia polypeptides, oligopeptides and proteins are included within thedefinition of peptide. In addition, peptides can be part of a (chimeric)protein or can be (part of) an RNA or DNA sequence encoding the peptideor protein.

[0026] Of course, functional derivatives as well as fragments of thepeptide according to the invention are also included in the presentinvention. Functional derivatives are meant to include peptides whichdiffer in one or more amino acids in the overall sequence, which havedeletions, substitutions, inversions or additions. Amino acidsubstitutions which can be expected not to essentially alter biologicaland immunological activities have been described. Amino acidreplacements between related amino acids or replacements which haveoccurred frequently in evolution are, inter alia Ser/Ala, Ser/Gly,Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M.D., Atlas of protein sequenceand structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5,suppl. 3). Based on this information, Lipman and Pearson developed amethod for rapid and sensitive protein comparison (Science 227,1435-1441, 1985) and determining the functional similarity betweenhomologous polypeptides.

[0027] Furthermore, as functional derivatives of these peptides are alsomeant to include other peptide-analogues derived from gp100 (or Melan)that are able to induce target cell lysis by tumor infiltratinglymphocytes.

[0028] In addition, with functional derivatives of these peptides arealso meant addition salts of the peptides, amides of the peptides andspecifically the C-terminal amides, esters and specifically theC-terminal esters and N-acyl derivatives specifically N-terminal acylderivatives and N-acetyl derivatives.

[0029] The peptides according to the invention can be producedsynthetically, by recombinant DNA technology or by viruses, if the aminoacid sequence of the peptide is encoded by a DNA sequence which is partof the virus DNA. Methods for producing synthetic peptides are wellknown in the art.

[0030] The organic chemical methods for peptide synthesis are consideredto include the coupling of the required amino acids by means of acondensation reaction, either in homogenous phase or with the aid of aso-called solid phase. The condensation reaction can be carried out asfollows:

[0031] condensation of a compound (amino acid, peptide) with a freecarboxyl group and protected other reactive groups with a compound(amino acid, peptide) with a free amino group and protected otherreactive groups, in the presence of a condensation agent;

[0032] condensation of a compound (amino acid, peptide) with anactivated carboxyl group and free or protected other reaction groupswith a compound (amino acid, peptide) with a free amino group and freeor protected other reactive groups.

[0033] Activation of the carboxyl group can take place, inter alia, byconverting the carboxyl group to an acid halide, azide, anhydride,imidazolide or an activated ester, such as the N-hydroxy-succinimide,N-hydroxy-benzotriazole or p-nitrophenyl ester.

[0034] The most common methods for the above condensation reactions are:the carbodiimide method, the azide method, the mixed anhydride methodand the method using activated esters, such as described in ThePeptides, Analysis, Synthesis, Biology Vol. 1-3 (Ed. Gross, E. andMeienhofer, J.) 1979, 1980, 1981 (Academic Press, Inc.).

[0035] Production of peptides by recombinant DNA techniques is a generalmethod which is known, but which has a lot of possibilities all leadingto somewhat different results. The polypeptide to be expressed is codedfor by a DNA sequence or more accurately by a nucleic acid sequence.

[0036] Also part of the invention is the nucleic acid sequencecomprising the sequence encoding the peptides according to the presentinvention.

[0037] Preferably, the sequence encoding the peptides according to thepresent invention are the sequences shown in SEQ ID NOS: 1-4 and 32-34.

[0038] As is well known in the art, the degeneracy of the genetic codepermits substitution of bases in a codon to result in another codonstill coding for the same amino acid, e.g., the codon for the amino acidglutamic acid is both GAT and GAA. Consequently, it is clear that forthe expression of a polypeptide with an amino acid sequence as shown inSEQ ID NO: 1-4, 9 or 32-34 use can be made of a derivate nucleic acidsequence with such an alternative codon composition thereby differentnucleic acid sequences can be found.

[0039] “Nucleotide sequence” as used herein refers to a polymeric formof nucleotides of any length, both to ribonucleic acid (RNA) sequencesand to deoxyribonucleic acid (DNA) sequences. In principle, this termrefers to the primary structure of the molecule. Thus, this termincludes double and single stranded DNA, as well as double and singlestranded RNA, and modifications thereof.

[0040] A further part of the invention are peptides, which areimmunogenic fragments of the peptide-analogues.

[0041] Immunogenic fragments are fragments which still have the abilityto induce an immunogenic response, i.e., that it is either possible toevoke antibodies recognizing the fragments specifically, or that it ispossible to find T lymphocytes which have been activated by thefragments. Another possibility is a DNA vaccine.

[0042] As has been said above, it has been known that the immunogenicaction of tumor associated antigens is often elicited through a T cellactivating mechanism (Townsend et al., 1989, H., Ann. Rev. Immunol. 7,601-624). Cytotoxic T lymphocytes (CTLs) recognizing melanoma cells in aT-cell receptor (TCR)-dependent and MHC-restricted manner have beenisolated from tumor-bearing patients (Knuth et al., 1992, Cancersurveys, 39-52). It has been shown that a peptide derived fromtyrosinase, another melanocyte-specific antigen, is recognized by a CTLclone (Brichard et al., 1993, J. Exp. Med., 178, 489-495).

[0043] It is known that the activation of T cells through the MHCmolecule necessitates processing of the antigen of which short pieces(for example 8-12 mers) are presented to the T lymphocyte.

[0044] Preferably, the peptides according to the present invention areflanked by non-related sequences, i.e., sequences with which they arenot connected in nature, because it has been found that such flankingenhances the immunogenic properties of these peptides, probably througha better processing and presentation by APCs.

[0045] Another part of the invention is formed by nucleotide sequencescomprising the nucleotide sequences coding for the above mentionedpeptides or an array of peptides.

[0046] Next to the use of these sequences for the production of thepeptides with recombinant DNA techniques, which will be exemplifiedfurther, the sequence information disclosed in the sequence listings forthe peptides according to the present invention can be used fordiagnostic purposes.

[0047] From these sequences primers can be derived as basis for adiagnostic test to detect gp100 or gp100-like proteins by a nucleic acidamplification technique for instance the polymerase chain reaction (PCR)or the nucleic acid sequence based amplification (NASBA) as described inU.S. Pat. No. 4,683,202 and EP 329,822, respectively.

[0048] These nucleotide sequences can be used for the production of thepeptides according to the present invention with recombinant DNAtechniques. For this, the nucleotide sequence must be comprised in acloning vehicle which can be used to transform or transfect a suitablehost cell.

[0049] A wide variety of host cell and cloning vehicle combinations maybe usefully employed in cloning the nucleic acid sequence. For example,useful cloning vehicles may include chromosomal, non-chromosomal andsynthetic DNA sequences such as various known bacterial plasmids, andwider host range plasmids such as pBR 322, the various pUC, pGEM andpBluescript plasmids, bacteriophages, e.g. lambda-gt-Wes, Charon 28 andthe M13 derived phages and vectors derived from combinations of plasmidsand phage or virus DNA, such as SV40, adenovirus or polyoma virus DNA(Rodriquez et al., 1988, ed. Vectors, Butterworths; Lenstra et al.,1990, Arch. Virol., 110, 1-24).

[0050] Useful hosts may include bacterial hosts, yeasts and other fungi,plant or animal hosts, such as Chinese Hamster Ovary (CHO) cells,melanoma cells, dendritic cells, monkey cells and other hosts.

[0051] Vehicles for use in expression of the peptides may furthercomprise control sequences operably linked to the nucleic acid sequencecoding for the peptide. Such control sequences generally comprise apromoter sequence and sequences which regulate and/or enhance expressionlevels. Furthermore, an origin of replication and/or a dominantselection marker are often present in such vehicles. Of course, controland other sequences can vary depending on the host cell selected.

[0052] Techniques for transforming or transfecting host cells are quiteknown in the art (for instance, Maniatis et al., 1982/1989, Molecularcloning: A laboratory Manual, Cold Spring Harbor Lab.).

[0053] It is extremely practical if, next to the information for thepeptide, also the host cell is co-transformed or co-transfected with avector which carries the information for an MHC molecule to which saidpeptide is known to bind. Preferably, the MHC molecule is HLA-A2.1,HLA-A1 or HLA-A3.1, or any other HLA allele which is known to be presentin melanoma patients. HLA-A2.1 is especially preferred because it hasbeen established (Anichini et al., 1993, J. Exp. Med., 177, 989-998)that melanoma cells carry antigens recognized by HLA-A2.1 restrictedcytotoxic T cell clones from melanoma patients.

[0054] Host cells especially suited for the expression of the peptidesaccording to the present invention are the murine EL4 and P8.15 cells.For expression of said peptides human BLM cells (Katano et al., 1984, J.Cancer Res. Clin. Oncol. 108, 197) are especially suited because theyalready are able to express the MHC molecule HLA-A2.1.

[0055] The peptides according to the present invention can be used in avaccine for the treatment of melanoma.

[0056] In addition to an immunogenically effective amount of the activepeptide, the vaccine may contain a pharmaceutically acceptable carrieror diluent.

[0057] The immunogenicity of the peptides of the invention, especiallythe oligopeptides, can be enhanced by cross-linking or by coupling to animmunogenic carrier molecule (i.e., a macromolecule having the propertyof independently eliciting an immunological response in a patient, towhich the peptides of the invention can be covalently linked) or if partof a protein.

[0058] Covalent coupling to the carrier molecule can be carried outusing methods well known in the art, the exact choice of which will bedictated by the nature of the carrier molecule used. When theimmunogenic carrier molecule is a protein, the peptides of the inventioncan be coupled, e.g., using water soluble carbodiimides such asdicyclohexylcarbodiimide, or glutaraldehyde.

[0059] Coupling agents such as these can also be used to cross-link thepeptides to themselves without the use of a separate carrier molecule.Such cross-linking into polypeptides or peptide aggregates can alsoincrease immunogenicity.

[0060] Examples of pharmaceutically acceptable carriers or diluentsuseful in the present invention include stabilizers such as SPGA,carbohydrates (e.g., mannose, sorbitol, mannitol, starch, sucrose,glucose, dextran), proteins such as albumin or casein, proteincontaining agents such as bovine serum or skimmed milk and buffers(e.g., phosphate buffer).

[0061] Optionally, one or more compounds having adjuvant activity may beadded to the vaccine. Suitable adjuvants are for example aluminiumhydroxide, phosphate or oxide, oil-emulsions (e.g. of Bayol F® or Marcol52®), saponins or vitamin-E solubilisate.

[0062] Dendritic cells are professional APC that express mannosereceptor used to take up antigen thus facilitating antigen processing.

[0063] The vaccine according to the present invention can be given interalia intravenously, intraperitoneally, intranasally, intradermally,subcutaneously or intramuscularly.

[0064] The useful effective amount to be administered will varydepending on the age and weight of the patient and mode ofadministration of the vaccine.

[0065] The vaccine can be employed to specifically obtain a T cellresponse, but it is also possible that a B cell response is elicitedafter vaccination. If so, the B cell response leads to the formation ofantibodies against the peptide of the vaccine, which antibodies will bedirected to the source of the antigen production, i.e., the tumor cells.This is an advantageous feature, because in this way the tumor cells arecombated by responses of both the immunological systems.

[0066] Both immunological systems will even be more effectivelytriggered when the vaccine comprises the peptides as presented in an MHCmolecule by an antigen presenting cell (APC). Antigen presentation canbe achieved by using monocytes, macrophages, interdigitating cells,Langerhans cells and especially dendritic cells, loaded with one of thepeptides of the invention or loading with protein including peptide ormanosylated protein. Loading of the APCs can be accomplished by bringingthe peptides of the invention into or in the neighborhood of the APC,but it is more preferable to let the APC process the complete gp100antigen. In this way a presentation is achieved which mimicks the invivo situation most realistically. Furthermore, the MHC used by the cellis of the type which is suited to present the epitope.

[0067] An overall advantage of using APCs for the presentation of theepitopes is the choice of APC cell that is used in this respect. It isknown from different types of APCs that there are stimulating APCs andinhibiting APCs.

[0068] Preferred APCs include, but are not limited to, the listed celltypes, which are so-called “professional” antigen presenting cells,characterized in that they have co-stimulating molecules, which have animportant function in the process of antigen presentation. Suchco-stimulating molecules are, for example, B7, CD25, CD40, CD70, CTLA-4or heat stable antigen (Schwartz, 1992, Cell 71, 1065-1068).

[0069] Fibroblasts, which have also been shown to be able to act as anantigen presenting cell, lack these co-stimulating molecules.

[0070] It is also possible to use cells already transfected with acloning vehicle harboring the information for the melanocyte peptideanalogues and which are cotransfected with a cloning vehicle whichcomprises the nucleotide sequence for an MHC class I molecule, forinstance the sequence coding for HLA A2.1, HLA A1 or HLA A3.1. Thesecells will act as an antigen presenting cell and will present peptideanalogues in the MHC class I molecules which are expressed on theirsurface. It is envisaged that this presentation will be enhanced, whenthe cell is also capable of expressing one of the above-mentionedco-stimulating molecules (in particular B7 (B7.1, B7.2), CD40), or amolecule with a similar function (e.g., cytokines transfected in cellline). This expression can be the result of transformation ortransfection of the cell with a third cloning vehicle having thesequence information coding for such a co-stimulating molecule, but itcan also be that the cell already was capable of production ofco-stimulating molecules.

[0071] Instead of a vaccine with these cells, which next to the desiredexpression products, also harbor many elements which are also expressedand which can negatively affect the desired immunogenic reaction of thecell, it is also possible that a vaccine is composed with liposomeswhich expose MHC molecules loaded with peptides, and which, forinstance, are filled with lymphokines. Such liposomes will trigger animmunologic T cell reaction.

[0072] By presenting the peptide in the same way as it is also presentedin vivo, an enhanced T cell response will be evoked. Furthermore, by thenatural adjuvant working of the relatively large, antigen presentingcells also a B cell response is triggered. This B cell response willalso lead to the formation of antibodies directed to the peptide-MHCcomplex. This complex is especially found in tumor cells, where it hasbeen shown that in the patient epitopes of gp100 are presentednaturally, which are thus able to elicit a T cell response. It is thisnaturally occurring phenomenon which is enlarged by the vaccination ofAPCs already presenting the peptides of the invention. By enlarging notonly an enlarged T cell response will be evoked, but also a B cellresponse which leads to antibodies directed to the MHC-peptide complexwill be initiated.

[0073] The vaccines according to the invention can be enriched bynumerous compounds which have an enhancing effect on the initiation andthe maintenance of both the T cell and the B cell response aftervaccination.

[0074] In this way, addition of cytokines to the vaccine will enhancethe T cell response. Suitable cytokines are for instance interleukins,such as IL-2, IL-4, IL-7, or IL-12, GM-CSF, RANTES, MIP-α, and tumornecrosis factor, and interferons, such as IFN- or the chemokins.

[0075] In a similar way, antibodies against T cell surface antigens,such as CD2, CD3, CD27 and CD28 will enhance the immunogenic reaction.

[0076] Also, the addition of helper epitopes to stimulate CD4⁺ helpercells or CD8⁺ killer cells augments the immunogenic reaction.Alternatively, also helper epitopes from other antigens can be used, forinstance from heat shock derived proteins or cholera toxin.

[0077] Another part of the invention is formed by using reactive tumorinfiltrating lymphocytes (TILs) directed against the peptides accordingto the present invention. In this method, the first step is taking asample from a patient. This is usually done by resection of a tumordeposit under local anesthesia. The TILs present in this specimen arethen expanded in culture for four to eight weeks, according to knownmethods (Topalian et al., 1987, J. Immunol. Meth. 102, 127-141). Duringthis culture, the TILs are then checked for reactivity with the peptidesaccording to the present invention or gp100-protein. The TILs whichrecognize the antigen are isolated and cultured further.

[0078] The reactive tumor infiltrating lymphocytes which are obtainedthrough this method, also form part of the invention. An example of suchTIL cell line, designated TIL 1200, has been found which specificallyreacts with gp100 and its epitopes. This TIL 1200 cell line alsoexpresses the MHC molecule HLA-A2.1. Furthermore, expression of TCR α/β,CD3 and CD8 by this cell line has been demonstrated. Furthermore, TIL1200 recognizes transfectants expressing both HLA-A2.1 and gp100.

[0079] TIL 1200 and other TILs recognizing gp100 are suited fortreatment of melanoma patients. For such treatment, TILs may be culturedas stated above, and they are given back to the patients by anintravenous infusion. The success of treatment can be enhanced bypre-treatment of the tumor bearing host with either total body radiationor treatment with cyclophosphamide and by the simultaneousadministration of interleukin-2 (Rosenberg et al., 1986, Science 223,1318-1321).

[0080] The TILs infused back to the patient are preferably autologousTILs (i.e., derived from the patient's own tumor) but also infusion withallogenic TILs can be imagined.

[0081] A further use of the TILs obtained by the method as describedabove is for in vivo diagnosis. Labeling of the TILs, for instance with¹¹¹In (Fisher et al., 1989, J. Clin. Oncol. 7, 250-261) or any othersuitable diagnostic marker, renders them suited for identification oftumor deposits in melanoma patients.

[0082] Another part of the invention is formed by the T cell receptor(TCR) expressed by reactive CTLs directed against the peptides accordingto this invention or the gp100-protein. As is well known in the art, theTCR determines the specificity of a CTL. Therefore, the cDNA encodingthe TCR, especially its variable region, can be isolated and introducedinto T cells, thereby transferring anti-tumor activity to any T cell.Especially introduction of such a TCR into autologous T cells andsubsequent expansion of these T cells will result in large numbers ofCTL suitable for adoptive transfer into the autologous patient.

[0083] Cells harboring this T cell receptor can also be used forvaccination purposes.

[0084] A vaccine can also be composed from melanoma cells capable ofexpression of the peptides according to the present invention. It ispossible to isolate these cells from a patient, using specificantibodies, such as NKI-beteb (directed against gp100), but is alsopossible to produce such melanoma cells from cultured melanoma celllines, which either are natural gp100-producers or have been manipulatedgenetically to produce the peptides according to the present invention.These cells can be irradiated to be non-tumorogenic and infused (back)into the patient. To enhance the immunologic effect of these melanomacells it is preferred to alter them genetically to produce a lymphokine,preferably interleukine-2 (IL-2) or granulocyte-macrophage colonystimulation factor (GM-CSF). Peptide⁺/gp100⁺ melanoma cells can betransfected with a cloning vehicle having the sequence coding for theproduction of IL-2 or GM-CSF.

[0085] Infusion of such a vaccine into a patient will stimulate theformation of CTLs.

[0086] Another type of vaccination having a similar effect isvaccination with pure DNA, for instance the DNA of a vector or a vectorvirus having the DNA sequence encoding the peptides according thepresent invention (both homologues and heterologues (chimeric protein)or repetitive). Once injected, the virus will infect or the DNA will betransformed to cells which express the antigen or the peptide(s).

[0087] Antibodies directed against the peptides according to the presentinvention are also part of the invention.

[0088] Monospecific antibodies to these peptides can be obtained byaffinity purification from polyspecific antisera by a modification ofthe method of Hall et al. (1984, Nature 311, 379-387). Polyspecificantisera can be obtained by immunizing rabbits according to standardimmunization schemes.

[0089] Monospecific antibody as used herein is defined as a singleantibody species or multiple antibody species with homogeneous bindingcharacteristics for the relevant antigen. Homogeneous binding as usedherein refers to the ability of the antibody species to bind to ligandbinding domain of the invention.

[0090] The antibody is preferably a monoclonal antibody, more preferablya humanized monoclonal antibody.

[0091] Monoclonal antibodies can be prepared by immunizing inbred mice,preferably Balb/c with the appropriate protein by techniques known inthe art (Köhler, G. and Milstein C., 1975, Nature 256, 495-497).Hybridoma cells are subsequently selected by growth in hypoxanthine,thymidine and aminopterin in an appropriate cell culture medium such asDulbecco's modified Eagle's medium (DMEM). Antibody producing hybridomasare cloned, preferably using the soft agar technique of MacPherson(1973, Tissue Culture Methods and Applications, Kruse and Paterson,eds., Academic Press). Discrete colonies are transferred into individualwells of culture plates for cultivation in an appropriate culturemedium. Antibody producing cells are identified by screening with theappropriate immunogen. Immunogen positive hybridoma cells are maintainedby techniques known in the art. Specific anti-monoclonal antibodies areproduced by cultivating the hybridomas in vitro or preparing ascitesfluid in mice following hybridoma injection by procedures known in theart.

[0092] It may be preferred to use humanized antibodies. Methods forhumanizing antibodies, such as CDR-grafting, are known (Jones et al.,1986, Nature 321, 522-525). Another possibility to avoid antigenicresponse to antibodies reactive with polypeptides according to theinvention is the use of human antibodies or fragments or derivativesthereof.

[0093] Human antibodies can be produced by in vitro stimulation ofisolated B-lymphocytes, or they can be isolated from (immortalized)B-lymphocytes which have been harvested from a human being immunizedwith at least one ligand binding domain according to the invention.

[0094] Antibodies as described above can be used for the passivevaccination of melanoma patients. A preferred type of antibodies forthis kind of vaccine are antibodies directed against the above-mentionedpeptides presented in connection with the MHC molecule. To produce thesekind of antibodies immunization of peptides presented by APCs isrequired. Such an immunization can be performed as described above.Alternatively, human antibodies to peptide-MHC complexes can be isolatedfrom patients treated with a vaccine consisting of APCs loaded with oneof said peptides.

[0095] The antibodies, which are formed after treatment with one of thevaccines of the invention can also be used for the monitoring of saidvaccination. For such a method, serum of the patients is obtained andthe antibodies directed to the peptide with which has been vaccinatedare detected. Knowing the antibody titre from this detection, it can bejudged if there is need for a boost vaccination.

[0096] Specific detection of said antibodies in the serum can beachieved by labeled peptides. The label can be any diagnostic markerknown in the field of in vitro diagnosis, but most preferred (and widelyused) are enzymes, dyes, metals and radionuclides, such as ⁶⁷Ga,^(99m)Tc, ¹¹¹In, ^(113m)In, ¹²³I, ¹²⁵I, or ¹³¹I.

[0097] The radiodiagnostic markers can be coupled directly to thepeptides of the invention or through chelating moieties which have beencoupled to the peptide directly or through linker or spacer molecules.The technique of coupling of radionuclides to peptides or peptide-likestructures is already known in the field of (tumor) diagnostics from thenumerous applications of labeled antibodies used both in in vivo and inin vitro tests.

[0098] Direct labeling of peptides can, for instance, be performed asdescribed in the one-vial method (Haisma et al., 1986, J. Nucl. Med. 27,1890). A general method for labeling of peptides through chelators, withor without linker or spacer molecules, has, for instance, been describedin U.S. Pat. Nos. 4,472,509 and 4,485,086. Chelators using a bicyclicanhydride of DTPA have been disclosed in Hnatowich et al. (1983, J.Immunol. Meth. 65, 147-157). Coupling through diamide dimercaptidecompounds has been disclosed in EP 188,256.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0099] The present invention is further described by way of exampleswith reference to the accompanying figures, in which:

[0100]FIG. 1. Target cell sensitization of alanine replacement epitopes.(A) Chromium labeled T2 target cells were preincubated for 1 hour withvarious amounts of the indicated alanine-substituted epitope-analogues.Melan-A/MART-1 27-35-reactive TIL 1235 lymphocytes were added at aneffector to target ratio of 20. (B) Target cell sensitization ofalanine-substituted gp100 154-162-analogues was analyzed usinggp100-reactive TIL 1200 lymphocytes at an effector to target ratio of20.

[0101]FIG. 2. Target cell sensitization of N-terminal anchor-replacementepitopes. Chromium release experiments were performed as in FIG. 1. (A)Melan-A/MART-1 27-35-reactive TIL 1235 lymphocytes were used to assaytarget cell sensitization by the Melan-A/MART-1 27-35 analogues. (B)Gp100 154-162-reactive TIL 1200 lymphocytes were used to assay targetcell sensitization by the gp100 154-162-analogues.

[0102]FIG. 3. Immunogenicity of gp100 154-162 epitope-analogues inHLA-A*0201/K^(b) transgenic mice. Bulk CTL obtained from immunized micewere tested for lytic activity using chromium labeled Jurkat A2/K^(b)target cells that were preincubated with no peptide, 10 mM wild typegp100 154-162 or 10 mM of the epitope-analogue used to immunize themice. For each peptide the mean specific lysis of bulk CTL of theresponding mice is shown. Standard deviations never exceeded 15% of themean value. One representative experiment out of two is shown.

[0103]FIG. 4. Peptide specific reactivity of in vitro inducedepitope-analogue specific CTL cultures. Chromium-labeled HLA-A*0201⁺ T2target cells were pre-incubated with 10 mM of an irrelevantHLA-A*0201-binding peptide, 10 mM wild type gp100 154-162 or 10 mM ofthe epitope-analogue used for CTL induction. The different CTL cultureswere added at an effector to target ratio of 20:1. One representativeexperiment out of two is shown.

[0104]FIG. 5. Epitope-analogue induced CTL cultures specifically lysemelanoma cells endogenously presenting the wild type epitope.Chromium-labeled HLA-A2.1⁺ BLM and Mel 624 melanoma cells were used astarget cells. BLM cells lack expression of gp100. The different CTLcultures were added at an effector to target ratio of 20:1. Onerepresentative experiment out of two is shown.

DETAILED DESCRIPTION OF THE INVENTION Materials and Methods

[0105] Cell Culture.

[0106] The HLA-A*0201⁺ melanoma line BLM was cultured as describedpreviously (Bakker et al., 1994, J. Exp. Med. 179:1005). TIL 1200 andTIL 1235 lymphocytes were cultured as was reported previously (Kawakamiet al., 1992, J. Immunol. 148:638). T2 cells (Salter et al., 1985,Immunogenetics. 21:235) and HLA-A*0201⁺ B lymphoblastoid JY cells weremaintained in Iscoves medium (Gibco, Paisley, Scotland UK) supplementedwith 5% FCS (BioWhittaker, Verviers, Belgium). Jurkat A*0201/K^(b) cells(Irwin et al., 1989, J. Exp. Med. 170:1091) expressing theHLA-A*0201/K^(b) chimeric molecule were cultured in Iscoves medium with5% FCS supplemented with 0.8 mg/ml G418 (Gibco, Paisley, Scotland UK).

[0107] HLA-A*0201⁺ Lymphocytes.

[0108] Healthy caucasian volunteers were phenotyped HLA-A2 by flowcytometry using mAbs BB7.2 (Parham et al., 1981, Hum. Immunol. 3:277)and MA2.1 (Parham et al., 1978, Nature 276:397). The donors underwentleukapheresis and PBMC were isolated by Ficoll/Hypaque density gradientcentrifugation. The cells were cryopreserved in aliquots of 4×10⁷ PBMC.

[0109] Transgenic Mice

[0110] HLA-A*0201/K^(b) transgenic mice were used (animal distributorHarlan Sprague Dawley, Inc., Indianapolis, USA). Mice were held underclean conventional conditions. The transgenic mice express the productof the HLA-A*0201/K^(b) chimeric gene in which the α3 domain of theheavy chain is replaced by the corresponding murine H-2 K^(b) domainwhile leaving the HLA-A*0201 α1 and α2 domains unaffected (Vitiello etal., 1991, J. Exp. Med. 1007). This allows the murine CD8 molecule onthe murine CD8⁺ T lymphocytes to interact with the syngeneic α3 domainof the hybrid MHC class I molecule.

[0111] Peptides.

[0112] For induction of CTL and chromium-release assays, peptides weresynthesized with a free carboxy-terminus by Fmoc peptide chemistry usingan ABIMED multiple synthesizer. All peptides were >90% pure as indicatedby analytical HPLC. Peptides were dissolved in DMSO and stored at −20°C.

[0113] HLA-A*0201 Upregulation On T2 Cells.

[0114] Peptide-induced HLA-A*0201 upregulation on T2 cells was performedas described previously (Nijman et al., 1993, Eur. J. Immunol. 23:1215).Briefly, peptides were diluted from DMSO stocks to variousconcentrations (final DMSO concentration 0.5%) and were incubatedtogether with 10⁵ T2 cells for 14 hours at 37° C., 5% CO₂ in serum-freeIscoves medium in a volume of 100 ml in the presence of 3 mg/ml humanβ2-microglobulin (Sigma, St Louis, Mo.). Stabilization of HLA-A*0201molecules at the cell surface of T2 cells was analyzed by flow cytometryusing anti-HLA-A2 mAb BB7.2 (Parham et al., 1981, Hum. Immunol. 3:277).The Fluorescence Index is expressed as: (experimental meanfluorescence÷background mean fluorescence)−1. The background meanfluorescence values were obtained by incubating T2 cells with aHLA-A*0201 non-binding peptide at similar concentrations.

[0115] Competition Based HLA-A*0201 Peptide-Binding Assay.

[0116] Peptide-binding to HLA-A*0201 was analyzed using HLA-A*0201⁺ JYcells as was described previously (van der Burg et al., 1995, Hum.Immunol. 44:189). Briefly, mild-acid treated JY cells were incubatedwith 150 nM Fluorescein (FL)-labeled reference peptide (FLPSDC(-FL)FPSV)and with several concentrations of competitor peptide for 24 hours at 4°in the presence of 1.0 mg/ml β2-microglobulin (Sigma, St. Louis, Mo.).Subsequently, the cells were washed, fixed with paraformaldehyde andanalyzed by flow cytometry. The mean-fluorescence (MF) obtained in theabsence of competitor peptide was regarded as maximal binding andequated to 0%; the MF obtained without reference peptide was equated to100% inhibition. % inhibition of binding was calculated using theformula: (1−(MF 150 nM reference & competitor peptide−MF no referencepeptide)÷(MF 150 nM reference peptide−MF no reference peptide))×100%.The binding capacity of competitor peptides is expressed as theconcentration needed to inhibit 50% of binding of the FL-labeledreference peptide (IC₅₀).

[0117] Measurement of MHC-Peptide Complex Stability at 37° C.

[0118] Measurement of MHC-peptide complex stability was performed.HLA-A*0201⁺ homozygous JY cells were treated with 10⁻⁴ M emetine (Sigma,St. Louis, USA) for 1 hour at 37° C. to stop de novo synthesis of MHCclass I molecules. The cells were then mild-acid treated andsubsequently loaded with 200 mM of peptide for 1 hour at roomtemperature. Thereafter, the cells were washed twice to remove freepeptide and were incubated at 37° C. for 0, 2, 4 and 6 hours.Subsequently, the cells were stained using mAb BB7.2 (Parham et al.,1981, Hum. Immunol. 3:277), fixed with paraformaldehyde and analyzed byflow cytometry.

[0119] CTL Induction in HLA-A*0201/K^(b) Transgenic Mice.

[0120] Groups of 3 HLA-A*0201/K^(b) transgenic mice were injectedsubcutaneously in the base of the tail vein with 100 mg peptideemulsified in IFA in the presence of 140 mg of the H-2I-A^(b)-restricted HBV core antigen-derived T helper epitope (128-140;sequence TPPAYRPPNAPIL) (Milich et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:1610). After 11 days, mice were sacrificed and spleen cells(30×10⁶ cells in 10 ml) were restimulated in vitro with peptide-loadedsyngeneic irradiated LPS-stimulated B cell lymphoblasts (ratio 4:1). Atday 6 of culture, the bulk responder populations were tested forspecific lytic activity.

[0121] HLA-A*0201⁺ Donor Derived CTL Induction in Vitro

[0122] Using thawed PBMC, dendritic cells were generated according theprocedure of Romani et al. (Romani et al., 1994, J. Exp. Med. 180:83) aswas described previously (Bakker et al., 1995, Cancer Res. 55:5330).Before the onset of culture, dendritic cells were loaded with 50 mM ofpeptide. Autologous CD8⁺ enriched responder T lymphocytes were preparedby adhering thawed PBMC for 2 hours and by subsequent partial depletionof the non-adherent fraction of CD4⁺ T cells using the anti-CD4 mAbRIV-7 (Leerling et al., 1990, Dev. Biol. Stand. 71:191) andSheep-anti-Mouse-IgG coated magnetic beads (Dynal, Oslo, Sweden). At theonset of stimulation, 2×10⁵ peptide-loaded DC and 2×10⁶ responder cellswere co-cultured per well of a 24-well tissue culture plate (Costar,Badhoevedorp, The Netherlands) in 2 ml of Iscoves medium containing 5%pooled human AB⁺ serum, 10³ U/ml IL-6 (Sandoz, Basel, Switzerland) and 5ng/ml IL-12.

[0123] On day 8 and day 15, the responder populations were restimulatedusing peptide-pulsed dendritic cells as stimulator cells. The cultureswere propagated in medium containing IL-2 (Cetus Corp., Emeryville,Calif.) and IL-7 (Genzyme, Cambridge, Mass.) at final concentrations of10 U/ml and 5 ng/ml respectively. Weekly hereafter the cultures wererestimulated using adherent peptide-pulsed PBMC as was describedpreviously (Bakker et al., 1995, Cancer Res. 55:5330). Responderpopulations were tested for specific lytic activity after at least 4rounds of restimulation.

[0124] Chromium-Release Assay.

[0125] Chromium release assays were performed as described previously(Bakker et al., 1994, J. Exp. Med. 179:1005.). Briefly, 10⁶ target cellswere incubated with 100 mCi Na₂ ⁵¹CrO₄ (Amersham, Bucks, UK) for 1 hour.Various amounts of effector cells were then added to the target cells intriplicate wells of U bottomed microtiter plates (Costar, Badhoevedorp,The Netherlands) in a final volume of 150 ml. In peptide recognitionassays, target cells were pre-incubated with various concentrations ofpeptide for 30 or 60 min at 37° C. in a volume of 100 ml prior to theaddition of effector cells. After 5 h of incubation, part of thesupernatant was harvested and its radioactive content was measured. Themean percentage specific lysis of triplicate wells was calculated usingthe formula: % specific lysis=((experimental release−spontaneousrelease)÷(maximal release−spontaneous release))×100.

EXAMPLE 1

[0126] Identification of Amino Acid Residues Engaged in HLA-A*0201Binding and/or TCR Interactions for the Melan-A/MART-1 27-35 and thegp100 154-162 Epitopes.

[0127] The Melan-A/MART-1 27-35 and the gp100 154-162 epitopes have beenidentified using HLA-A*0201 restricted TIL lines derived from metastaticmelanomas. The Melan-A/MART-1 27-35 epitope was found to be the nominalepitope capable of triggering the Melan-A/MART-1 specific TIL 1235 linewhen presented on HLA-A*0201⁺ target cells (Kawakami et al., 1994. J.Exp. Med. 180:347). Among a panel of peptides ranging from 8-mers to11-mers located around gp100 amino acids 155-161, we identified the9-mer 154-162 as the peptide most efficient in sensitizing HLA-A*0201⁺target cells for lysis by the gp100 reactive TIL 1200 line (Bakker etal., 1995, Int. J. Cancer 62:97). Both the Melan-A/MART-1 27-35 9-merand the gp100 154-162 9-mer have now been eluted from the cell surfaceof HLA-A*0201⁺ melanoma cells, and were identified by tandemmass-spectroscopy, indicating that they are indeed the nominal epitopesendogenously presented in HLA-A*0201. To identify amino acid residues inboth epitopes engaged in HLA-A*0201 binding and/or TCR interactions,epitope-analogues were synthesized in which the native amino acid wasreplaced by an alanine residue. In case alanine residues were present inthe wild type epitope, they were substituted for the amino acid glycine.The substituted peptides were assayed for binding to HLA-A*0201 by meansof an indirect binding assay using the processing defective cell line T2(Nijman et al., 1993, Eur. J. Immunol. 23:1215). All substitutions inthe Melan-A/MART-1 epitope resulted in a nearly complete loss in thecapability to stabilize HLA-A*0201 molecules at the cell surface of T2cells (Table I). When the Melan-A/MART-1 27-35 analogues were used atmicromolar concentrations to sensitize HLA-A*0201⁺ target cells forlysis by Melan-A/MART-1-specific CTL, we observed a decrease in targetcell lysis for the alanine replacements at positions 4 to 7 of theepitope (Table I). In addition, the glycine substitution at position 2resulted in decreased CTL reactivity. The amino acids at these positionsin the Melan-A/MART-1 27-35 epitope are therefore most likely involvedin TCR interactions.

[0128] In case of the gp100 154-162 epitope decreased HLA-A*0201affinity of epitope-analogues was only observed for the alaninesubstitutions at position 3 and 9 (Table 1). With respect to T cellrecognition, alanine substitutions at positions 5, 6 and 7 of theepitope were not allowed, indicating that amino acids at these positionsare critical contact residues within this epitope for the TCR.

[0129] Subsequently, the epitope-analogues that induced reactivity atmicromolar concentrations were titrated to evaluate their relativeability to sensitize T2 target cells for lysis by the relevant CTL (FIG.1). In all cases the epitope-analogues were similar or inferior comparedto the wild type epitope in their sensitizing capacity, except for thealanine substitution at position 8 of the gp100 154-162 epitope.Surprisingly, this peptide was able to induce target cell lysis bygp100-reactive CTL even at a tenfold lower concentration.

EXAMPLE 2

[0130] N-Terminal Anchor Residue Replacements in Both the gp100 154-162and the Melan-A/MART-1 27-35 Epitopes Result in Improved Affinity forHLA-A*0201.

[0131] Since both the Melan-A/MART-1 27-35 and the gp100 154-162epitopes have non-conventional N-terminal anchoring residues, wereplaced these residues for the common HLA-A*0201 anchoring residues V,L, I or M (Drijfhout et al., 1995, Hum. Immunol. 43:1). Subsequently, wetested these peptides for HLA-A*0201 binding and their ability tosensitize target cells for lysis by the relevant CTL. Apart from themethionine substitution, all anchor residue replacements in theMelan-A/MART-1 epitope resulted in significantly improved binding toHLA-A*0201 (Table II). HLA-A*0201⁺ target cells loaded with thesepeptides at a concentration of 1 mM were recognized by theMelan-A/MART-1 reactive CTL, except for the methionine substitutedepitope (Table II). Although this peptide did bind to HLA-A*0201 at alevel comparable to the wild type epitope, it failed to induce CTLreactivity. Titration experiments using the Melan-A/MART-1 anchorreplacement peptides demonstrated that these epitope-analogues wereinferior to wild type in sensitizing target cells for lysis by TIL 1235(FIG. 2).

[0132] Using the T2 assay all gp100 154-162 anchor replacement peptidesexcept the methionine substituted epitope showed HLA-A*0201 bindingcomparable to the wild type epitope (Table II). Interestingly, thesepeptides were recognized by TIL 1200 when loaded on target cells attenfold lower concentrations compared to the wild type peptide (FIG. 2),while the methionine substituted peptide showed no difference. Thesefindings demonstrate that amino acid substitutions within the nativeepitope can result in improved T cell recognition.

EXAMPLE 3

[0133] Improved Target Cell Sensitization by gp100 154-162 EpitopeAnalogues Correlates With Increased Affinity for HLA-A*0201.

[0134] To assess whether the augmented CTL recognition of thesubstituted gp100 154-162 epitopes could be attributed to improvedHLA-A*0201 affinity, the HLA-A*0201 binding capacity of these peptideswas tested now using a more sensitive cell-bound HLA-A*0201 bindingassay based on competition of a labeled reference peptide with thepeptides of interest (van der Burg et al., 1995, Hum. Immunol. 44:189).HLA-A*0201 binding-affinities obtained with this assay demonstrated thatall peptides that were able to sensitize target cells for lysis by TIL1200 at tenfold lower concentrations compared to wild type, also boundwith higher affinity to HLA-A*0201 (Table III). In addition to theN-terminal anchor substitutions, replacement of a polar residue for ahydrophobic residue adjacent to the C-terminal anchoring position alsoresulted in an epitope-analogue with improved HLA-A*0201 affinity(KTWGQYWAV (SEQ ID NO: 1)), apparently without affecting TCRrecognition. Measurement of MHC class I-peptide complex dissociationrates demonstrated that the epitope-analogues tested are at leastequally stable when compared to wild type (Table III). All peptidestested showed a DT₅₀ (the time required for 50% of the complexes todecay) longer than 4 hours. Peptides with DT₅₀ values of≧3 hours wereimmunogenic in HLA-A*0201/K^(b) transgenic mice. Taken together, thesedata indicate that the gp100 154-162 epitope-analogues may have similaror increased immunogenicity compared to wild type gp100 154-162.

EXAMPLE 4

[0135] Immunogenicity of gp100 154-162 Epitope-Analogues inHLA-A*0201/K^(b) Transgenic Mice.

[0136] In order to determine the in vivo immunogenicity of the gp100154-162 epitope-analogues of which the MHC class I binding-affinity anddissociation rate was measured. HLA-A*0201/K^(b) transgenic mice werevaccinated with the gp100 154-162 wild type epitope, with theepitope-analogues KTWGQYWAV (SEQ ID NO: 1), KVWGQYWQV (SEQ ID NO: 2),KLWGQYWQV (SEQ ID NO: 3) or KIWGQYWQV (SEQ ID NO: 4), or with a controlpeptide (HBV core 18-27: FLPSDDFPSV (SEQ ID NO: 6)). The generation ofthese transgenic mice (Vitiello et al., 1991. J. Exp. Med. 173:1007) andtheir use to analyze in vivo immunogenicity have been describedpreviously (Ressing et al., 1995, J. Immunol. 154:5934; Sette et al.,1994, J. Immunol. 153:5586). As shown in FIG. 3, the gp100 154-162epitope-analogues KTWGQYWAV (SEQ ID NO: 1), KVWGQYWQV (SEQ ID NO: 2),and KLWGQYWQV (SEQ ID NO: 3), very efficiently induced a CTL response.To a lesser extent also the epitope-analogue KIWGQYWQV (SEQ ID NO: 4)and the wild type gp100 154-162 were able to elicit a CTL response. BulkCTL derived from mice vaccinated with the gp100 154-162epitope-analogues specifically lysed Jurkat A*0201/K^(b) cells loadedwith both the peptide used for vaccination and the wild type epitope.Interestingly, CTL bulk cultures raised against the epitope-analoguesall recognized target cells pulsed with the wild type epitope equallywell or better compared to target cells pulsed with epitope-analoguesused for vaccination. Thus, all gp100 154-162 epitope-analogues testedwere immunogenic in HLA-A*0201/K^(b) transgenic mice, and elicited CTLdisplaying cross-reactivity with the native gp100 154-162 epitope.

EXAMPLE 5

[0137] In Vitro Induction of gp100 154-162 Epitope-Analogue SpecificHuman CTL Displaying Cross-Reactivity With Endogenously HLA-A*0201Presented Wild Type gp100 154-162.

[0138] Next, we performed in vitro CTL induction assays to assesswhether within the T cell repertoire of HLA-A*0201⁺ healthy donorsprecursor T lymphocytes were present capable of recognizing gp100154-162 epitope-analogues. In order to achieve this, we initiatedcultures of peptide-loaded dendritic cells together with autologousresponder T lymphocytes as described previously (Bakker et al., 1995,Cancer Res. 55:5330). After several rounds of restimulation, responder Tcells were tested for cytotoxic activity (FIG. 4). All bulk CTLpopulations raised against the gp100 154-162 epitope-analogues,KTWGQYWAV (SEQ ID NO: 1), KVWGQYWQV (SEQ ID NO: 2), KLWGQYWQV (SEQ IDNO: 3) and KIWGQYWQV (SEQ ID NO: 4), efficiently lysed HLA-A*0201⁺ T2target cells incubated with the peptides used for CTL induction. Onlylow background lysis was observed in the presence of an irrelevantpeptide. In addition, these gp100 154-162 epitope-analogue reactive CTLefficiently lysed T2 target cells incubated with wild type gp100154-162. To address the question whether these CTL responder populationscould also recognize endogenously processed and presented wild typeepitope, we performed chromium-release experiments using HLA-A*0201⁺melanoma cell lines BLM and Mel 624 as targets. BLM cells have lostexpression of the gp100 antigen, both at the protein and at the mRNAlevel (Adema et al., 1993, Am. J. Pathol. 143:1579). As shown in FIG. 5,all peptide-induced CTL cultures lysed the antigen expressing Mel 624cells, whereas no or background lysis was observed against antigennegative BLM cells. TNF release by the anti-gp100 154-162 analogue CTLfurther demonstrated the reactivity of these CTL with endogenouslypresented wild type gp100 154-162 (data not shown). These data show thatthe four different CTL cultures induced using gp100 154-162epitope-analogue loaded dendritic cells, all recognized the native gp100154-162 epitope endogenously processed and presented by HLA-A*0201⁺ Mel624 cells. TABLE I HLA-A*0201-binding and target cell sensitization ofalanine-replacement epitopes. target target cell cell HLA-A*0201 lysisby HLA-A*0201 lysis stabilization^(a) TIL stabilization by TIL MelanA/MART-1 27-35 50 μM 25 μM 1235^(b) gp100 154-162 50 μM 25 μM 1200YLEPGPVTA^(c) (SEQ ID NO: 7) 2.26 2.12 −3 YLEPGPVTA (SEQ ID NO: 7) 3AAGIGILTV (SEQ ID NO: 8) 1.20 1.11 40 KTWGQYWQV (SEQ ID NO: 6) 2.06 1.4067 GAGIGILTV (SEQ ID NO: 10) 1.07 1.11 52 ATWGQYWQV (SEQ ID NO: 11) 1.941.42 75 AGGIGILTV (SEQ ID NO: 12) 0.96 1.05 6 KAWGQYWQV (SEQ ID NO: 13)1.57 1.20 64 AAAIGILTV (SEQ ID NO: 14) 0.98 0.99 13 KTAGQYWQV (SEQ IDNO: 15) 1.17 1.02 58 AAGAGILTV (SEQ ID NO: 16) 0.93 0.97 0 KTWAQYWQV(SEQ ID NO: 17) 1.45 1.13 63 AAGIAILTV (SEQ ID NO: 18) 1.01 1.01 4KTWGAYWQV (SEQ ID NO: 19) 1.59 1.25 9 AAGIGALTV (SEQ ID NO: 20) 0.931.00 2 KTWGQAWQV (SEQ ID NO: 21) 1.42 1.15 7 AAGIGIATV (SEQ ID NO: 22)1.10 1.13 6 KTWGQYAQV (SEQ ID NO: 23) 1.31 1.14 −2 AAGIGILAV (SEQ ID NO:24) 1.05 1.01 11 KTWGQYWAV (SEQ ID NO: 1) 1.72 1.35 73 AAGIGILTA (SEQ IDNO: 25) 1.00 1.03 26 KTWGQYWQA (SEQ ID NO: 26) 1.08 1.02 76

[0139] TABLE II HLA-A*0201-binding and target cell sensitization ofN-terminal anchor-replacement epitopes. HLA- HLA-A*0201 A*0201stabilization^(a) target cell stabilization target cell 50 25 lysis byTIL 50 25 lysis by TIL Melan A/MART-1 27-35 μM μM 1235^(b) gp100 154-162μM μM 1200 YLEPGPVTA^(c) (SEQ ID NO: 7) 2.26 2.12 −1 YLEPGPVTA (SEQ IDNO: 7) 3 AAGIGILTV (SEQ ID NO: 8) 1.20 1.11 40 KTWGQYWQV (SEQ ID NO: 9)2.06 1.40 67 AVGIGILTV (SEQ ID NO: 27) 1.62 1.36 27 KVWGQYWQV (SEQ IDNO: 2) 2.13 1.57 69 ALGIGILTV (SEQ ID NO: 28) 2.21 1.93 16 KLWGQYWQV(SEQ ID NO: 3) 2.19 1.55 65 AMGIGILTV (SEQ ID NO: 29) 1.18 1.05 6KMWGQYWQV (SEQ ID NO: 35) 1.73 1.28 57 AIGIGLTV (SEQ ID NO: 30) 1.581.29 27 KIWGQYWQV (SEQ ID NO: 4) 2.00 1.43 68

[0140] TABLE III HLA-A*0201 binding and complex stability of gp100154-162 epitope-analogues Affinity Stability peptide IC50 (μM)^(a) (DT50%)^(b) FLPSDFFPSV^(c) (SEQ ID NO: 31) 0.5 >4 hr KTWGQYWQV (SEQ ID NO:9) 1.4 >4 hr KTWGQYWAV (SEQ ID NO: 1) 0.5 >4 hr KVWGQYWQV (SEQ ID NO: 2)0.8 >4 hr KLWGQYWQV (SEQ ID NO: 3) 0.4 >4 hr KIWGQYWQV (SEQ ID NO: 4)0.6 >4 hr

[0141]

1 35 1 9 PRT epitope analogue 1 Lys Thr Trp Gly Gln Tyr Trp Ala Val 1 52 9 PRT epitope analogue 2 Lys Val Trp Gly Gln Tyr Trp Gln Val 1 5 3 9PRT epitope analogue 3 Lys Leu Trp Gly Gln Tyr Trp Gln Val 1 5 4 9 PRTepitope analogue 4 Lys Ile Trp Gly Gln Tyr Trp Gln Val 1 5 5 13 PRT Thelper epitope 5 Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu 1 510 6 10 PRT control peptide 6 Phe Leu Pro Ser Asp Asp Phe Pro Ser Val 15 10 7 9 PRT alanine-replacement epitope 7 Tyr Leu Glu Pro Gly Pro ValThr Ala 1 5 8 9 PRT alanine-replacement epitope 8 Ala Ala Gly Ile GlyIle Leu Thr Val 1 5 9 9 PRT alanine-replacement epitope 9 Lys Thr TrpGly Gln Tyr Trp Gln Val 1 5 10 9 PRT alanine-replacement epitope 10 GlyAla Gly Ile Gly Ile Leu Thr Val 1 5 11 9 PRT alanine-replacement epitope11 Ala Thr Trp Gly Gln Tyr Trp Gln Val 1 5 12 9 PRT alanine-replacementepitope 12 Ala Gly Gly Ile Gly Ile Leu Thr Val 1 5 13 9 PRTalanine-replacement epitope 13 Lys Ala Trp Gly Gln Tyr Trp Gln Val 1 514 9 PRT alanine-replacement epitope 14 Ala Ala Ala Ile Gly Ile Leu ThrVal 1 5 15 9 PRT alanine-replacement epitope 15 Lys Thr Ala Gly Gln TyrTrp Gln Val 1 5 16 9 PRT alanine-replacement epitope 16 Ala Ala Gly AlaGly Ile Leu Thr Val 1 5 17 9 PRT alanine-replacement epitope 17 Lys ThrTrp Ala Gln Tyr Trp Gln Val 1 5 18 9 PRT alanine-replacement epitope 18Ala Ala Gly Ile Ala Ile Leu Thr Val 1 5 19 9 PRT alanine-replacementepitope 19 Lys Thr Trp Gly Ala Tyr Trp Gln Val 1 5 20 9 PRTalanine-replacement epitope 20 Ala Ala Gly Ile Gly Ala Leu Thr Val 1 521 9 PRT alanine-replacement epitope 21 Lys Thr Trp Gly Gln Ala Trp GlnVal 1 5 22 9 PRT alanine-replacement epitope 22 Ala Ala Gly Ile Gly IleAla Thr Val 1 5 23 9 PRT alanine-replacement epitope 23 Lys Thr Trp GlyGln Tyr Ala Gln Val 1 5 24 9 PRT alanine-replacement epitope 24 Ala AlaGly Ile Gly Ile Leu Ala Val 1 5 25 9 PRT alanine-replacement epitope 25Ala Ala Gly Ile Gly Ile Leu Thr Ala 1 5 26 9 PRT alanine-replacementepitope 26 Lys Thr Trp Gly Gln Tyr Trp Gln Ala 1 5 27 9 PRTanchor-replacement epitope 27 Ala Val Gly Ile Gly Ile Leu Thr Val 1 5 289 PRT anchor-replacement epitope 28 Ala Leu Gly Ile Gly Ile Leu Thr Val1 5 29 9 PRT anchor-replacement epitope 29 Ala Met Gly Ile Gly Ile LeuThr Val 1 5 30 8 PRT anchor-replacement epitope 30 Ala Ile Gly Ile GlyLeu Thr Val 1 5 31 10 PRT epitope-analogue 31 Phe Leu Pro Ser Asp PhePhe Pro Ser Val 1 5 10 32 9 PRT epitope analogue 32 Lys Ile Trp Gly GlnTyr Trp Ala Val 1 5 33 9 PRT epitope analogue 33 Lys Leu Trp Gly Gln TyrTrp Ala Val 1 5 34 9 PRT epitope analogue 34 Lys Val Trp Gly Gln Tyr TrpAla Val 1 5 35 9 PRT epitope analogue 35 Lys Met Trp Gly Gln Tyr Trp GlnVal 1 5

What is claimed is:
 1. A peptide comprising: at least part of the aminoacid sequence of SEQ ID NO:9 wherein an original amino acid at position2 thereof is substituted with valine and wherein said peptide isimmunogenic with lymphocytes directed against metastatic melanomas. 2.The peptide of claim 1, wherein the original amino acid at position 8,glutamine, is substituted by replacement amino acid, alanine.
 3. Thepeptide of claim 2, wherein the original amino acid at position 2,threonine, is substituted by a replacement amino acid selected from thegroup consisting of isoleucine, leucine, and valine.
 4. The peptide ofclaim 1, wherein the peptide comprises the amino acid sequence of SEQ IDNO:
 1. 5. A nucleic acid which comprises a nucleotide sequence encodingthe peptide of claim
 1. 6. A vaccine comprising the peptide of claim 1or a nucleotide sequence encoding said peptide.
 7. The vaccine of claim6, further comprising a pharmaceutically acceptable carrier or diluent.8. The vaccine of claim 6, wherein an antigen presenting cell has beenpreloaded with the peptide.
 9. A vaccine which comprises a T cellreceptor against the peptide of claim 1 or cells expressing said T cellreceptor.
 10. The vaccine of claim 6, characterized in that it alsocomprises one or more compounds selected from the group consisting of anadjuvant, one or more cytokines, antibodies directed against CD2, CD3,CD27, CD28 or other T cell surface antigens and helper epitopes tostimulate CD4+ or CD8+ T cells.
 11. The vaccine of claim 9,characterized in that it also comprises one or more compounds selectedfrom the group consisting of an adjuvant, one or more cytokines,antibodies directed against CD2, CD3, CD27, CD28 or other T cell surfaceantigens and helper epitopes to stimulate CD4+ or CD8+ T cells
 12. Tumorinfiltrating lymphocytes which bind to the peptide of claim
 1. 13. Avaccine comprising the tumor infiltrating lymphocytes of claim
 13. 14.An antibody directed against the peptide of claim
 1. 15. A compositioncomprising the antibody of claim
 14. 16. A method for monitoringprogress of immunotherapy in a subject, the method comprising: detectingthe presence of antibodies directed to the peptide of claim 1 in theserum of the subject as indicative of the progress of immunotherapy.